Further information has been obtained regarding conditions favoring dissociation of the oxysterol-binding protein complex into its lowest molecular weight form (Mr = 97,000). An unidentified component present in whole cytosol promotes complete dissociation of the 196 kilodalton form of the complex at pH 7.4 within 18 hrs at 0~C to give the lower molecular weight form. This factor, which does not seem to be a protease, is largely, but not completely, eliminated from binding protein preparations partially purified by ammonium sulfate precipitation. It appears to be relatively abundant in mouse liver and hepatoma as compared to adult brain. We have developed three mutant Chinese hamster ovary cell lines that are resistant to both compactin (a competitive repressor of HMG-CoA reductase) and 25-hydroxycholesterol (a repressor of the synthesis of the reductase). These mutant lines exhibit defective binding of 25-hydroxycholesterol to the oxysterol binding protein when intact cells are treated with the sterol. However, the binding protein can be identified in a partially purified fraction of the cytosol. We think that these mutant lines will be useful in establishing the role of the binding protein in regulations of sterol synthesis. We have now identified small amounts of two sterol fractions in extracts of Chinese hamster lung cells that repress HMG-CoA reductase in cell cultures. One of these has relatively low affinity for the binding protein. This sterol migrates with 24,25-epoxycholesterol on reverse phase and micro Porasil columns. It has been shown that this epoxide can arise from squalene 2,3;22,23-diepoxide in cells wherein squalene metabolism has been blocked. The second sterol band appears to migrate with 25-hydroxycholesterol. The identities of both of these sterols have, however, not been established. We have synthesized 5-alpha-cholesta 1,8(14), 9(11)-triene-3,15 dione. After reduction of the 2(3) bond with tritium and enzymatic reduction of the 3-ketone we expect that this compound will serve as an effective photoaffinity label for the binding protein. (D)